ABSTRACT
The tegument protein pp150 of Human Cytomegalovirus (HCMV) is known to be essential for the final stages of virus maturation and mediates its functions by interacting with capsid proteins. Our laboratory has previously identified the critical regions in pp150 important for pp150-capsid interactions and designed peptides similar in sequence to these regions, with a goal to competitively inhibit capsid maturation. Treatment with a specific peptide (PepCR2 or P10) targeted to pp150 conserved region 2 led to a significant reduction in murine CMV (MCMV) growth in cell culture, paving the way for in vivo testing in a mouse model of CMV infection. However, the general pharmacokinetic parameters of peptides, including rapid degradation and limited tissue and cell membrane permeability, pose a challenge to their successful use in vivo. Therefore, we designed a biopolymer-stabilized elastin-like polypeptide (ELP) fusion construct (ELP-P10) to enhance the bioavailability of P10. Antiviral efficacy and cytotoxic effects of ELP-P10 were studied in cell culture, and pharmacokinetics, biodistribution, and antiviral efficacy were studied in a mouse model of CMV infection. ELP-P10 maintained significant antiviral activity in cell culture, and this conjugation significantly enhanced P10 bioavailability in mouse tissues. The fluorescently labeled ELP-P10 accumulated to higher levels in mouse liver and kidneys as compared to the unconjugated P10. Moreover, viral titers from vital organs of MCMV-infected mice indicated a significant reduction of virus load upon ELP-P10 treatment. Therefore, ELP-P10 has the potential to be developed into an effective antiviral against CMV infection.
Subject(s)
Antiviral Agents , Cytomegalovirus Infections , Elastin , Muromegalovirus , Peptides , Phosphoproteins , Viral Matrix Proteins , Animals , Elastin/chemistry , Elastin/metabolism , Cytomegalovirus Infections/drug therapy , Cytomegalovirus Infections/virology , Mice , Antiviral Agents/pharmacology , Antiviral Agents/pharmacokinetics , Antiviral Agents/chemistry , Peptides/pharmacology , Peptides/chemistry , Muromegalovirus/drug effects , Humans , Capsid Proteins/metabolism , Capsid Proteins/chemistry , Cytomegalovirus/drug effects , Capsid/metabolism , Capsid/drug effects , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/pharmacokinetics , Disease Models, Animal , Elastin-Like PolypeptidesABSTRACT
Geometries of Early Pleistocene [2.58 to 0.78 million years (Ma) ago] ice sheets in northwest Europe are poorly constrained but are required to improve our understanding of past ocean-atmosphere-cryosphere coupling. Ice sheets are believed to have changed in their response to orbital forcing, becoming, from about 1.2 Ma ago, volumetrically larger and longer-lived. We present a multiproxy data set for the North Sea, extending to over a kilometer below the present-day seafloor, which demonstrates spatially extensive glaciation of the basin from the earliest Pleistocene. Ice sheets repeatedly entered the North Sea, south of 60°N, in water depths of up to ~250 m from 2.53 Ma ago and subsequently grounded in the center of the basin, in deeper water, from 1.87 Ma ago. Despite lower global ice volumes, these ice sheets were near comparable in spatial extent to those of the Middle and Late Pleistocene but possibly thinner and moving over slippery (low basal resistance) beds.
ABSTRACT
Porcine dermal collagen permanently crosslinked with hexamethylene diisocyanate was investigated for its suitability as a dermal tissue engineering matrix. It was found that the chemically crosslinked collagen had far fewer free lysine groups per collagen molecule than did the uncrosslinked matrix. The ability of the matrix to support human primary fibroblast outgrowth from explants was compared for matrices that had been presoaked in various solutions, including fibroblast media, cysteine and phosphate buffered saline (PBS). It was found that superior cell outgrowth was obtained after soaking with fibroblast media and PBS. The fibroblast attachment properties of the matrix were compared against tissue culture plastic and PET. The collagen matrix showed the least amount of cell retention compared to the other to matrices, however, the general trends were similar for all three scaffolds. Longer term cultures on the collagen showed fibroblasts covering the matrix stacking up on each other and bridging natural hair follicles. However, it was also observed that the fibroblasts were not able to penetrate into the matrix structure. This was believed to result from the chemical crosslinking, as shown by the resistance of the matrix to degradation by collagenases.
